The objectives of these studies is to analyze the regulatory mechanisms implicated in antibody-dependent cell-mediated cytotoxicity (ADCC) and, specifically, the determinant peptide sequence of the FcgammaRI involved in the regulation of the receptor in monocytes and cells of the monocytic lineage. To study the nature of putative cytolytic molecules involved in ADCC by monocytic effectors, cells were induced to differentiate with either gamma-IFN or PMA, and new protein synthesis was inhibited at various time points with cycloheximide. Inhibition of protein synthesis with cycloheximide during the early stages of differentiation, resulted in diminution of ADCC against antibody-coated SRBC. Partially or completely differentiated cells were resistant to the effect of protein synthesis inhibition. Unstimulated U937 cells demonstrated FcgammaRI, II and III surface receptors, but were unable to mediate cytotoxicity. Stimulation with gamma-IFN increased FcRI expression and allowed the cells to mediate ADCC. Surface FcgammaRI expression was not increased when cells were induced with PMA. Nevertheless, U937 cells were able to exert cytotoxicity against antibody-coated SRBC. These results provide evidence that in the monocyte linage, two dissociable components are implicated in the antibody-mediated cytotoxic mechanism against SRBC: the high affinity receptor for immunoglobulines (FcgammaRI), and an inducible protein-based lytic mechanism. These two elements developed independently during "in vitro" differentiation, as suggested by the difference in their respective induction time. The inability of cycloheximide to block ADCC in more differentiated cells, suggests that preformed lytic molecules mediate cytotoxicity and that FcgammaR mediated signals activate them without the need for new protein synthesis. We have also developed a retroviral construct encoding for the FcgammaRI. We are testing several cell lines(FcgammaRI negative/ADCC positive) to be use as targets for transfection. These will permit further study of FcRI-mediated ADCC, by deletion and/or point mutation of specific amino acid sequences to determine the sites involved in intracellular signaling.